非洲猪瘟病毒p72蛋白抗体全自动化学发光酶免疫检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.03.034

Abstract

Abstract:

Antibody detection is important for African swine fever virus (ASFV) infection diagnosis and vaccine research. Therefore, it is of great significance to establish a fully automatic chemiluminescent enzyme immunoassay (CLEIA) based on ASFV p72 protein antibody. For the detection, antibodies against p72 were captured by magnetic particles (MPs) coupled with purified p72 protein, then combined with alkaline phosphates (AP)-labelled p72, and ASFV p72 antibodies can be determined using a fully automated chemiluminescence analyzer. Under optimal working conditions, the cut-off value of the established CLEIA was 9.505 U·mL^-1 with a diagnostic specificity and sensitivity of 98.33% and 100%, respectively, and no cross-reactivity with other porcine pathogen-positive sera were found. The intra- and inter-batch coefficients of variation were less than 5% and 10%, respectively, and all components can be stored for more than 6 months at 4 ℃. The lowest detection limit for ASFV-positive standard serum was 1∶12 800. The total coincidence rate with the commercial kit was up to 95.90%, indicating a good applicability. In conclusion, the established CLIEA has the advantages of high sensitivity and reproducibility, combining with automated detection and simple operation steps, which can provide an important tool for the clinical diagnosis of ASF.

Key words: African swine fever virus, p72, chemiluminescent enzyme immunoassay, antibody detection

CLC Number:

S852.659.1

MA Xiaoli, LI Duan, ZENG Daoping, LIU Yanling, WANG Xiaomin, PENG Guoliang, SONG Changxu, WANG Lei, XU Zheng. Establishment of a Fully Automated Chemiluminescent Enzyme Immunoassay for Detecting Antibodies against African Swine Fever Virus p72[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1355-1365.

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References 31

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项目 Item	p72-MP用量/μL p72-MP volume
项目 Item	50	100	150	200
阳性血清RLU值(P) RLU value of positive serum (P)	167 220	238 945	225 090	240 024
阴性血清RLU值(N) RLU value of negative serum (N)	45 620	46 805	57 009	63 726
P/N值P/N value	3.66	5.11	3.95	3.77

项目 Item	p72-AP用量/μL p72-AP volume
项目 Item	50	100	150	200
阳性血清RLU值(P) RLU value of positive serum (P)	17 200	240 050	250 740	260 575
阴性血清RLU值(N) RLU value of negative serum (N)	41 201	42 575	55 099	59 366
P/N值P/N value	4.18	5.64	4.55	4.39

项目 Item	反应时间/min Reaction time
项目 Item	1	3	5	8
阳性血清RLU值(P) RLU value of positive serum (P)	120 056	175 578	273 667	269 900
阴性血清RLU值(N) RLU value of Negative serum (N)	30 093	41 173	45 552	45 892
P/N值P/N value	3.99	4.26	6.01	5.88

样品 Sample	稀释度 Dilution ratios	CLEIA		英吉钠ELISA		普泰ELISA
样品 Sample	稀释度 Dilution ratios	浓度	结果 Result	X/%	结果 Result	S/P	结果 Result
ASFV阳性标准血清 ASFV standard serum	1∶400	320.00	+	76	+	2.80	+
	1∶800	160.0	+	53	+	2.10	+
	1∶1 600	80.0	+	27	－	1.55	+
	1∶3 200	40.0	+	10	－	1.01	+
	1∶6 400	20.0	+	-7	－	0.59	+
	1∶12 800	10.0	+	-19	－	0.33	－
	1∶25 600	5.0	－	-21	－	0.21	－
临床样品 Clinical sample	1∶2	18.2	+	39	－	1.76	+
	1∶4	11.2	+	18	－	0.58	+
	1∶8	7.9	－	7	－	0.25	－
	1∶16	4.82	－	-2	－	0.19	－

血清编号 Serum number	批内变异系数Intrabatch coefficient of variation		批间变异系数Interbatch coefficient of variation
血清编号 Serum number	${\bar x}$±s	CV/%	${\bar x}$±s	CV/%
1	4.36±0.33	0.76	4.70±0.24	5.10
2	4.66±0.10	2.15	4.59±0.16	3.49
3	11.14±0.54	4.74	11.89±0.59	9.75
4	16.57±0.48	2.90	16.94±1.04	6.13
5	67.17±0.87	1.30	64.07±1.59	2.49
6	102.02±5.0	4.90	11.85±0.15	1.27

Establishment of a Fully Automated Chemiluminescent Enzyme Immunoassay for Detecting Antibodies against African Swine Fever Virus p72

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方法 Methods		商品化ELISA Commercial ELISA			符合率/% Coincidence rate
方法 Methods		阳性样品数 Positive serum numbers	阴性样品数 Negative serum numbers	总数 Total	符合率/% Coincidence rate
CLEIA	阳性样品数Positive serum numbers	15	3	18	83.33
	阴性样品数Negative serum numbers	2	102	104	98.10
	总数Total	17	105	122	95.90

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